Plasma long-chain fatty acids are the primary fuel source for energy production in the normal heart. In the ischemic and reperfused myocardium, there is decreased utilization of fatty acids. Furthermore, perfusion with fatty acids results in in situ membrane damage and cardiac dysfunction. Fatty acid-induced injury to ischemic myocardium can be reduced by inhibition of outer carnitine paImitoyltransferase (CPTO), a rate-limiting enzyme in fatty acid oxidation. Therefore, for effective pharmacotherapy of defects in cardiac fatty acid oxidation, it is important to understand the biochemical and molecular mechanisms regulating CPT. The mitochondrial membrane contains two distinct CPTs: an outer malonyl CoA-sensitive enzyme (CPTO) and an inner malonyl CoA- insensitive enzyme (CPTi). Membrane-bound CPTO is inhibited by malonyl CoA, and detergent solubilization of the mitochondrial membrane abolishes malonyl CoA sensitivity of the enzyme. Our working hypothesis, supported by reconstitution studies, is that CPTi and CPTO are identical enzymes or isoforms with common structural features, and malonyl CoA regulation of CPTO is provided by a separate malonyl CoA binding protein (MCBP). The specific aims of this proposal are: l. To clone and sequence a cDNA encoding the heart mitochondrial MCBP with the aid of monospecific polyclonaI antibodies and oligonucleotide probes constructed from partial sequences of the purified protein. 2. To express the cloned cDNA in E. coli or yeast cells to produce active MCBP as measured by (a) direct determination of native malonyl CoA- sensitive CPT activity, (b) conferral of malonyl CoA sensitivity to CPTi (an insensitive inner enzyme) by the expressed MCBP in a reconstituted system, and (c) malonyl CoA binding in control and transformed cells. 3. To map the malonyl CoA binding residues in the receptor using photolabeled radioactive malonyl CoA and determine the amino acid residue(s) that are essential for malonyl CoA binding by site-directed mutagenesis. The long-term objectives are to elucidate the regulatory mechanism of fatty acid transport and oxidation in the normal and ischemic heart.